Summary: Inflammatory immune responses with clinically relevant consequences can be associated with TNF, IL-1, IL-6 and other cytokines. These cytokines are rapidly produced by bacteria and bacterial components, such as DNA and LPS, but can also be induced, under certain circumstances, by triggering of complement receptors. Manufacture of plasma derivatives involves steps which are not aseptic and which can lead to bacterial contamination. Intact bacteria are removed by sterile filtration, but this step does not remove LPS, bacterial DNA (bDNA), or other microbial constituents. Adverse events with IGIV administration include fever and hypotension; fever has been associated with elevated TNF levels in recipients, and hypotension is a known side effect of LPS. We sought to determine whether the pattern of cytokine secretion by human monocytes was similar after IGIV and microbial stimulation of cells, and whether low levels of LPS and bDNA present in IGIV were sufficient to stimulate cytokine release. Multiple lots of IGIV from different manufacturers, including manufacturers of VIGIV, were assessed to determine whether they could stimulate TNF or IL-1 release from human mononuclear cells at concentrations that are present in humans after IGIV infusion. Many lots stimulated significant TNF and IL-1 production, most consistently observed when non-heat inactivated human serum was used in the culture media. Addition of polymyxin B, which neutralizes LPS, did not always abrogate cytokine release. Although no IGIV lots had LPS contamination > 0.91 EU/ml (the industry standard), dose response experiments using LPS showed that amounts lower than 0.91 EU/ml can stimulate cytokine release from monocytes. Bacterial DNA was detected in IGIV using PCR primers which recognized broadly conserved bacterial DNA sequences; the levels detected were lower than those needed to stimulate monocyte cytokines in vitro. However, the presence of bDNA suggests that other bacterial constituents, such as cell wall components of gram positive bacteria, could also be present, some of which may not be detected by current lot release standards such as LPS testing and rabbit pyrogen testing. In a related project, we are determining in vivo correlates of stimulation by bacterial components in relation to triggering of dendritic cell migration and activation in animal models. Recent results show that in the presence of (complement-component-containing) non-heat inactivated serum, proinflammatory cytokine release is greater, suggesting that complement components interact with IGIV, to produce this effect. We are focusing upon assays to detect, and methods to block this interaction. In the past year, we have also changed the tissue culture system from individual donor monocytes, to monocyte pools, derived from up to 10 donors. This has enabled us to accomplish many experiments using the same monocytes. We have found that certain products are more likely to stimulate cytokine release than others, and confirmed that these effects cannot be completely attributed to LPS. Finally, we have obtained cell lines that express particular toll-like receptors (TLR's), and express IL2 receptor on their cell surface, upon triggering of TLR's. These cell lines will be used to determine whether binding of these pattern-recognition receptors occurs when IGIV triggers cytokine release. This would further help to identify the agents in IGIV which mediate such effect, and could lead to development of better predictors of adverse events. Overall, these studies will add to understanding of how infusion-related IGIV adverse events are mediated, and may lead to manufacturing improvements which can abrogate such effects from all immune globulins.